S collected and immediately centrifuged. Title Loaded From File Plasma aliquots were sampled and stored at 280uC before measurement of adiponectin. Adipose tissue was dissected and immediately frozen in liquid 10781694 nitrogen and then stored at 280uC until analysis of 15d-PGJ3.Analysis of 15d-PGJ3 by Gas Chromatography (GC)-mass Spectrometry (GC-MS) and GC/tandem MS15d-PGJ3 putative compound eluting as a single peak from HPLC was collected. The compound was converted to a pentafluorobenzyl ester derivative, with pentafluorobenzyl bromide and diisopropylethylamide in acetonitrile. A Thermo Trace GC connected to a Thermo PolarisQ MS operated in negative ion chemical ionization (NICI) mode was used to analyze the sample. 15d-PGJ3 was detected by GC-MS using selected ion monitoring for the [M-CH2C6F5]- ion (m/z 313). The molecular ion m/z 313 of putative 15d-PGJ3 was subjected to collision-induced dissociation (CID).Analysis of 15d-PGJ3 and d5-15d-PGJ3 in the Culture Medium of 3T3-L1 Cells Incubated with EPA or d5-EPACells were incubated with EPA or d5-EPA. Products were separated by HPLC; the HPLC fraction supposed to contain 15dPGJ3 or d5-15d-PGJ3 was collected and was analyzed by GC-MS and GC-MS/MS as described above. The molecular ions m/z 313 of putative 15d-PGJ3 and m/z 318 of putative d5-15d-PGJ3 were subjected to collision-induced dissociation (CID). Spectra that are shown were obtained at 2 eV. Quantifications were performed using d4-15d-PGJ2.Dehydration/isomerization of PGD2 and PGDPGD2 or PGD3 (1 mM) were incubated in PBS at 37uC. After acidification to pH 3, samples were extracted three times with 4 volumes of ethyl acetate and extracts were dried under nitrogen gas. The samples were reconstituted in ethanol.EPA-Derived Prostaglandin and Adiponectinwith 1 mM or 10 mM EPA complexed with bovine serum albumin. Erin mRNA and the phosphorylation of Ecadherin were determined in BGC Adiponectin in the medium was determined by ELISA. Results are means 6 sem (n = 4 in triplicate), expressed as percentage of the control ( ). Statistical significance is represented as *P,0.05 vs control. doi:10.1371/journal.pone.0063997.gAnalysis of 15d-PGJ3 in Adipose Tissue from EPA FedmiceEpididymal adipose tissues were obtained from previously mentioned feeding study. Sample was extracted using a silica Sep-Pak cartridge. 15d-PGJ3 was purified by HPLC as described above, converted into a pentafluorobenzyl ester derivative, and analyzed by GC-MS/MS as described above.Statistical AnalysisStatistical analyses were performed using Student’s t-test. The difference was considered significant at p,0.05. The results are expressed as means 6 sem.Results Increased Plasma and Media Adiponectin Concentrations in Mice Fed an EPA-enriched Diet and in 3T3-L1 Cells Treated with EPA, RespectivelyWe examined the effects of EPA on the production of adiponectin in mice fed an EPA-enriched diet. Plasma level of adiponectin was significantly increased (+17 ) as early as 4 days after initiation of the EPA-enriched diet group compared to control mice (Fig. 2 A). On the contrary, blood leptin secretion was significantly decreased (by 1.5-fold) after 4 days of the EPA-enriched diet feeding (not shown). We also observed that the body weight gain of mice fed the EPA-enriched diet was significantly lower than that of mice fed the standard diet (31.0 g +/21.4 vs 33.9+/20.7) (Fig. 2 B). We also examined the effects of EPA on the adiponectin concentration in the culture media of cells. 3T3-L1 cells were incubated for 2 h or 4 h with 1 mM or 10 mM of EPA. As shown in Fig. 2 C, EPA i.S collected and immediately centrifuged. Plasma aliquots were sampled and stored at 280uC before measurement of adiponectin. Adipose tissue was dissected and immediately frozen in liquid 10781694 nitrogen and then stored at 280uC until analysis of 15d-PGJ3.Analysis of 15d-PGJ3 by Gas Chromatography (GC)-mass Spectrometry (GC-MS) and GC/tandem MS15d-PGJ3 putative compound eluting as a single peak from HPLC was collected. The compound was converted to a pentafluorobenzyl ester derivative, with pentafluorobenzyl bromide and diisopropylethylamide in acetonitrile. A Thermo Trace GC connected to a Thermo PolarisQ MS operated in negative ion chemical ionization (NICI) mode was used to analyze the sample. 15d-PGJ3 was detected by GC-MS using selected ion monitoring for the [M-CH2C6F5]- ion (m/z 313). The molecular ion m/z 313 of putative 15d-PGJ3 was subjected to collision-induced dissociation (CID).Analysis of 15d-PGJ3 and d5-15d-PGJ3 in the Culture Medium of 3T3-L1 Cells Incubated with EPA or d5-EPACells were incubated with EPA or d5-EPA. Products were separated by HPLC; the HPLC fraction supposed to contain 15dPGJ3 or d5-15d-PGJ3 was collected and was analyzed by GC-MS and GC-MS/MS as described above. The molecular ions m/z 313 of putative 15d-PGJ3 and m/z 318 of putative d5-15d-PGJ3 were subjected to collision-induced dissociation (CID). Spectra that are shown were obtained at 2 eV. Quantifications were performed using d4-15d-PGJ2.Dehydration/isomerization of PGD2 and PGDPGD2 or PGD3 (1 mM) were incubated in PBS at 37uC. After acidification to pH 3, samples were extracted three times with 4 volumes of ethyl acetate and extracts were dried under nitrogen gas. The samples were reconstituted in ethanol.EPA-Derived Prostaglandin and Adiponectinwith 1 mM or 10 mM EPA complexed with bovine serum albumin. Adiponectin in the medium was determined by ELISA. Results are means 6 sem (n = 4 in triplicate), expressed as percentage of the control ( ). Statistical significance is represented as *P,0.05 vs control. doi:10.1371/journal.pone.0063997.gAnalysis of 15d-PGJ3 in Adipose Tissue from EPA FedmiceEpididymal adipose tissues were obtained from previously mentioned feeding study. Sample was extracted using a silica Sep-Pak cartridge. 15d-PGJ3 was purified by HPLC as described above, converted into a pentafluorobenzyl ester derivative, and analyzed by GC-MS/MS as described above.Statistical AnalysisStatistical analyses were performed using Student’s t-test. The difference was considered significant at p,0.05. The results are expressed as means 6 sem.Results Increased Plasma and Media Adiponectin Concentrations in Mice Fed an EPA-enriched Diet and in 3T3-L1 Cells Treated with EPA, RespectivelyWe examined the effects of EPA on the production of adiponectin in mice fed an EPA-enriched diet. Plasma level of adiponectin was significantly increased (+17 ) as early as 4 days after initiation of the EPA-enriched diet group compared to control mice (Fig. 2 A). On the contrary, blood leptin secretion was significantly decreased (by 1.5-fold) after 4 days of the EPA-enriched diet feeding (not shown). We also observed that the body weight gain of mice fed the EPA-enriched diet was significantly lower than that of mice fed the standard diet (31.0 g +/21.4 vs 33.9+/20.7) (Fig. 2 B). We also examined the effects of EPA on the adiponectin concentration in the culture media of cells. 3T3-L1 cells were incubated for 2 h or 4 h with 1 mM or 10 mM of EPA. As shown in Fig. 2 C, EPA i.